Process of preparing etoposide phosphate and etoposide

ABSTRACT

Etoposide phosphate is prepared by coupling dibenzyl 4&#39;-demethyl-4-epipodophyllotoxin-4&#39;-phosphate with 2,3-di-O-benzyl-4,6-O-ethylidene-α,β-D-glucopyranose in a solvent and subsequently removing the protecting groups. The tetra-benzyl protected etoposide phosphate is recrystallized from methanol or directly crystallized from acetonitrile by adding methanol to yield substantially the pure C-1&#34;-β form. The benzyl protecting groups are simultaneously removed by hydrogenation to produce etoposide phosphate in high yields. In a further embodiment, etoposide phosphate is treated with a phosphatase enzyme to yield etoposide.

FIELD OF THE INVENTION

The present invention relates to a novel process for the preparation ofantitumor compounds, and to the novel intermediates for preparing theantitumor compounds. More particularly, the invention is directed to aprocess for preparing 4'-demethylepipodophyllotoxin glucoside4'-phosphates and the intermediate compounds for preparing thephosphates. The invention is particularly directed to a process forpreparing etoposide phosphate and for preparing etoposide from etoposidephosphate.

BACKGROUND OF THE INVENTION

Etoposide and teniposide are 4'-demethylepipodophyllotoxin glucosidederivatives which are widely used in clinical therapy for treatingcancer. In particular, etoposide is approved in the United States fortreating small cell lung cancer and testicular cancer. However,etoposide exhibits limited solubility in water which makes it difficultto formulate into suitable pharmaceutical compositions.

To increase the water solubility of etoposide and its ability to beadministered, etoposide phosphate is prepared as a prodrug. Etoposidephosphate metabolizes within the body to etoposide which can then beutilized by the body. One example of a water soluble prodrug isdescribed in U.S. Pat. No. 4,904,768 which discloses water solubleprodrugs of 4'-demethylepipodophyllotoxin glucoside derivatives bearinga 4'-phosphate group. One example disclosed therein is etoposide4'-phosphate. Etoposide 4'-phosphate is prepared by reacting etoposidewith phosphorous oxychloride followed by hydrolysis, or by reactingetoposide with diphenyl chlorophosphate followed by hydrogenation toremove the phenyl groups.

The preparation of epipodophyllotoxin glycosides are also disclosed inU.S. Pat. No. 4,997,931. The 4'-demethylepipodophyllotoxin glycosidesare prepared by condensing 4'-protected 4'-demethylepipodophyllotoxinwith a protected sugar. The resulting compound is then derivatized toproduce the corresponding 4'-phosphate.

The previous processes for preparing etoposide and etoposide phosphatetypically require the protection of the phenol, coupling with aprotected sugar and then the removal of the protecting groups. Inaddition, most of these methods require different protecting groups forthe hydroxy and phosphate groups. The different protecting groupsrequire multiple steps to remove respective protecting groups. Thedeprotection steps often require acid or alkaline conditions, which candegrade the final product, resulting in low yields.

Etoposide phosphate is usually prepared from etoposide by the additionalsteps of phosphorylation and deprotection. These multiple stepstypically result in lower overall yields of the desired compounds aswell as the expense and difficulty of producing the compounds due toundesirable phosphorylation of the glucosidic hydroxyls on etoposide.

SUMMARY OF THE INVENTION

The present invention is directed to a process of preparing4'-demethylepipodophyllotoxin glucoside 4'-phosphates, and in particularetoposide phosphate by coupling a novel protected sugar with a novelprotected 4'-demethyl-4-epipodophyllotoxin-4'-phosphate. Moreparticularly, the invention is directed to a di(arylmethyl)-protectedsugar and a tetra-arylmethyl-protected 4'-demethyl-4-epipodophyllotoxinglucoside 4'-phosphate and a process of preparing etoposide phosphatetherefrom. The arylmethyl protecting groups on the hydroxyl andphosphate groups may be the same or different, and are preferably benzylor benzyl substituted with one or more selected from the groupconsisting of C₁₋₄ alkyl, hydroxy, phenyl, benzyl, halogen, alkoxy,nitro and carboxylic acids and esters thereof. The di(arylmethyl)4'-demethyl-4-epipodophyllotoxin-4'-phosphate is prepared by reactingthe phenol with a di(arylmethyl) phosphite, a tetrahalomethane, atertiary amine, and an acylation catalyst in a suitable solvent.

The protected sugar according to one embodiment of the invention is2,3-di-O-benzyl-4,6-O-ethylidene-α,β-D-glucopyranose which is coupledwith dibenzyl 4'-demethyl-4-epipodophyllotoxin phosphate in a solvent toproduce the tetra benzyl protected etoposide phosphate. The protectedetoposide 4'-phosphate is crystallized or recrystallized to recover theC-1"-β anomer. The protecting groups are removed simultaneously from theglycosidic and phosphate groups by hydrogenating or other suitable meansto yield etoposide phosphate.

The overall process is efficient to yield etoposide 4'-phosphate in pureform without extensive purification steps. The protecteddibenzyl-4-(2,3-di-O-benzyl-4,6-O-ethylidine-β-D-glucopyranosyl)-4'-dimethyl-4-epipodophyllotoxin-4'-phosphateis easily crystallized from the reaction medium or recrystallized toisolate the C-1"-β form in substantially pure form. Isolation of thedesired anomer is usually obtained in a single crystallization step.

Specifically, the invention is directed to a process for preparing acompound having the Formula Va ##STR1## which comprises reacting acompound of Formula IIIb ##STR2## wherein R₁ is an arylmethyl hydroxyprotecting group and R₂ is arylmethyl or the two R₂ groups together areC₁₋₅ alkylidene, with a compound of Formula II in a reaction medium inthe presence of a Lewis acid ##STR3## where R₃ is arylmethyl and whereR₁, R₂ and R₃ are the same or different, to form the compound of FormulaIVb ##STR4## selectively crystallizing the C-1"-β anomer of Compound IVband subsequently removing the hydroxy and phosphate protecting group,and in cases where R₂ is an arylmethyl hydroxy protecting group,reacting Compound IVb with a carbonyl having one to five carbon atoms oran acetal equivalent thereof.

A further aspect of the invention is a process of preparing a compoundhaving the Formula VI ##STR5## which comprises reacting a compound ofFormula V in a buffer solution ##STR6## with a phosphatase enzyme toremove the phosphate, and recovering said compound of Formula VI.

Another aspect of the invention is to provide a process for preparing acompound of Formula VI ##STR7## comprising phosphorylating a compound ofFormula I ##STR8## with a phosphorylating agent to produce a protected4'-demethyl-4-epipodophyllotoxin-4'-phosphate of Formula II ##STR9##where R₃ is arylmethyl, reacting said compound of Formula II with aprotected sugar of Formula III ##STR10## to produce a compound ofFormula IV ##STR11## where R₁ is an arylmethyl protecting group;isolating the C-1"-β form of Formula IV; removing the hydroxy andphosphate protecting groups to produce a compound of Formula V ##STR12##and treating said compound of Formula V with a phosphatase enzyme toremove the phosphate group and produce the compound of Formula VI.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to an improved process for preparing4'-demethylepipodophyllotoxin glucoside 4'-phosphates and in particularetoposide 4'-phopshate, pharmaceutically acceptable salts, and solvatesthereof. The invention is further directed to the preparation ofarylmethyl protected sugars and arylmethyl protected precursors toetoposide and etoposide 4'-phosphate. The invention is further directedto a process of producing etoposide phosphate using hydroxy andphosphate protecting groups which allow easy separation of anomers bycrystallization. A further advantageous feature is the ease by which thehydroxy and phosphate protecting groups can be removed simultaneouslywithout degradation of the final product.

The process of the invention yields arylmethyl and, in particular,benzyl protected etoposide 4'-phosphate in a manner which can be easilyseparated to the anomerically pure C-1"-β form by crystallization fromthe reaction medium or by recrystallization from a suitable solvent. Theoverall process is rapid and efficient, providing an effective processfor preparing etoposide 4'-phosphate. The phosphate group can be easilyremoved by a phosphatase enzyme providing an efficient process forpreparing etoposide, pharmaceutically acceptable salts and solvatesthereof.

The overall process is efficient for producing etoposide phosphate oretoposide as discussed in greater detail hereinafter. In a preferredembodiment, dibenzyl 4'-demethyl-4-epipodophyllotoxin-4,-phosphate iscoupled in the presence of a Lewis acid with2,3-dibenzyl-4,6-O-ethylidene-α,β-D-glucopyranose to produce an anomericmixture of dibenzyl4-(2,3-di-O-benzyl-4,6-O-ethylidene-α,β-D-glucopyranosyl)-4-demethyl-4-epipodophyllotoxin-4'-phosphate.The C-1"-β anomer has surprisingly been found to easily crystallize fromsolution in substantially pure form. The C-1"-β anomer may be directlycrystallized from the reaction medium or recrystallized from a suitablesolvent. The C-1"-β anomer is then recovered and hydrogenated tosimultaneously remove the hydroxy and phosphate protecting groups.

As used herein, the term pharmaceutically acceptable salts include mono-and di-alkali metal salts, and alkaline earth metal salts. In preferredembodiments, the final compound is an ethanolate solvate. Solvates areformed by crystallization or recrystallization from organic solventssuch as ethanol using standard procedures. The term alkylidene includesstraight or branched alkyl chains including, for example, ethylidene,propylidene and isopropylidene.

In one aspect of the invention, the process leads to phosphorylation of4'-demethylepipodophyllotoxin of Formula I to produce a protecteddi(arylmethyl) 4'-demethylepipodophyllotoxin-4'-phosphate of Formula II.The phosphorylation process is preferably carried out by reacting4'-demethylepipodophyllotoxin with di(arylmethyl) phosphite, atetrahalomethane, a tertiary amine, and an acylation catalyst. Thetetrahalomethane has the formula CX₄ where X is a halogen selected fromthe group consisting of F, Cl, Br and I. In preferred embodiments, thetetrahalomethane is CCl₄. The halogens on the carbon may be the same ordifferent. The tertiary amine in preferred embodiments isN,N-diisopropylethylamine (DIPA), although other suitable tertiaryamines may be used. The acylation catalyst may be a standard catalyst asknown in the art. In preferred embodiments, the acylation catalyst isN,N-dimethylaminopyridine (DMAP). The reaction can be summarized asfollows: ##STR13## where R₃ is arylmethyl. In preferred embodiments, R₃is benzyl whereby the resulting phosphate has the structure of CompoundIIa. ##STR14## Alternatively, R₃ is a benzyl group substituted with oneor more selected from the group consisting of C₁₋₄ alkyl, hydroxy,phenyl, benzyl, halogen, alkoxy, nitro and carboxylic acids and estersthereof. Suitable substituted benzyl groups include, for example,2-methyl benzyl, 3-methyl benzyl, 4-methyl benzyl, 1 or 2-naphthyl, 2, 3or 4-phenyl benzyl, 4-methoxycarbonyl benzyl, 2,6-dichlorobenzyl,2-fluorobenzyl and pentafluorobenzyl.

This phosphorylation process is a convenient and easy process whichproduces the protected di(arylmethyl)4'-demethylepipodophyllotoxin-4'-phosphate in high yield. The process isessentially a one pot process that is rapid and highly selective to thephenolic hydroxy group of Compound I. Although the process isparticularly advantageous for the phosphorylation of4'-demethylepipodophyllotoxin, the process is general and highlyselective to phenols including, for example, p-fluorophenol,2,6-dimethoxyphenol, 1,2-benzenediol and 4-hydroxyphenethyl alcohol. Theprocess using 4-hydroxyphenethyl alcohol produced very littlephosphorylation at the primary alcohol. Phosphorylation of etoposidegave the desired product with less glycosidic phosphorylation than withthe preformed dibenzyl chlorophosphate.

The preferred solvent is acetonitrile, although any halogenated ornon-halogenated solvent may be used in the phosphorylation. Thetetrahalomethane and in particular carbon tetrachloride preferably isused only in reagent amounts rather than as a solvent as in someconventional processes. The amount of the tetrahalomethane used in thephosphorylation reaction is one or more equivalents per equivalent ofthe starting phenol. The reaction is also carried out under mildconditions at or below room temperature and typically below about -10°C. The phosphorylation reaction is further carried out substantially inthe absence of added dibenzylchlorophosphate (DBPCl) since DBPCl isgenerated in situ. This avoids the need to prepare DBPCl in a separatestep and reduces the impurity content of the resulting phosphorylatedproduct. Typically, the reaction proceeds to completion in about 45minutes. Compound II is recovered by standard methods such asrecrystallizing in isopropyl alcohol.

Compound II is then coupled with a hydroxy protected glucopyranose inthe presence of a Lewis acid. In preferred embodiments, the Lewis acidis boron trifluoride etherate. Alternative Lewis acids include, forexample, AlCl₃, ZnCl₂, Et₂ AlCl, CF₃ SO₃ H, CF₃ SO₃ Ag, Zn(CF₃ SO₃)₂ andTMSCF₃ SO₃. The coupling reaction may be carried out in the presence ofmolecular sieves. The coupling reaction is carried out in a halogenatedor non-halogenated solvent, most preferably acetonitrile. Other solventsinclude, for example, propionitrile, acetone, methylene chloride,chloroform, 1,2-dichloroethane and mixtures thereof.

A preferred hydroxy protected glucopyranose has the Formula III##STR15## where R₁ is arylmethyl. In preferred embodiments, R₁ is benzylsuch that the glucopyranose has the structure IIIa. ##STR16##

In further embodiments, R₁ is a substituted benzyl that is substitutedwith one or more selected from the group consisting of C₁₋₄ alkyl,hydroxy, phenyl, benzyl, halogen such as fluoro, chloro, bromo and iodo,alkoxy, nitro and carboxylic acids and esters thereof. Suitablesubstituted benzyl groups include 2-methyl benzyl, 3-methyl benzyl,4-methyl benzyl, 1 or 2-naphthyl, 2, 3 or 4-phenyl benzyl, 4-methoxycarbonyl benzyl, 2,6-dichlorobenzyl, 2-fluorobenzyl andpentafluorobenzyl. Typically, R₁ is the same as R₃.

The glucopyranose may further have the structure of Compound IIIb##STR17## where R₁ is as above and R₂ is the same as R₁, or the two R₂groups taken together are a C₁₋₅ alkylidene group. Preferably, the twoR₂ groups together are ethylidene. In alternative embodiments, the twoR₂ groups together may be propylidene or isopropylidene.

Compounds III, IIIa and IIIb are prepared by known procedures such asthat described in U.S. Pat. No. 4,997,931. The aryl protectedglucopyranose is formed as an anomeric mixture of C-1-α,β. Unlike mostanomeric mixtures, the C-1-β anomer of the arylmethyl glucopyranose canbe separated from the α anomer by crystallization. Specifically, theanomeric mixture of the glucopyranose Compound IIIa can be crystallizedfrom hexane to afford substantially anomerically pure C-1-β form ofCompound IIIa. Furthermore, the glucopyranose Compound IIIa having aninitial β:α composition of 1:1 solidifies over time to a ratio of 85:15β:α.

The coupling reaction of the protected glucopyranose of Compound IIIwith the protected 4'-demethyl-4-epipodophyllotoxin-4'-phosphate ofCompound II preferably is carried out in acetonitrile in the presence ofa Lewis acid. ##STR18##

The coupling of the hydroxy protected glucopyranose of Compound IIIawith the dibenzyl 4'-demethyl-4-epipodophyllotoxin-4'-phosphate (IIa)produces a C-1"-α,β anomeric mixture of dibenzyl4-(2,3-di-O-benzyl-4,6-O-ethylidene-D-glucopyranosyl)-4'-demethyl-4-epipodophyllotoxin-4'-phosphatehaving the Formula IVa. ##STR19##

The coupling reaction proceeds rapidly and easily in the presence ofboron trifluoride etherate to yield the α and β anomers of Compound IVa.

It is not necessary to isolate the D form of Compound III andparticularly IIIa prior to the coupling. The final ratio of IVa α andIVa β does not depend on the anomeric composition of the startingCompound IIIa when the reaction is carried out in halogenated solvents.In acetonitrile, the coupling of Compounds IIa and IIIa (85:15 β:α) inthe presence of boron trifluoride etherate at -20° C gives Compounds IVaβ and IVa α in a ratio of 72:28. It is believed that the anomerizationof the sugar occurs very rapidly in halogenated solvents, whileanomerization is much slower in acetonitrile.

In further embodiments, a suitable salt may be added to the reactionmixture to increase the ionic strength of the solvent. Suitable saltsinclude alkali and alkaline earth metal perchlorates. For example, theuse of 0.5M LiClO₄ dissolved in acetonitrile increased the ratio of IVaβ:α to 81:19.

The resulting anomeric mixture of Compound IVa α,β can be recrystallizedfrom methanol to obtain substantially the pure C-1"-β form in highyields. A single crystallization in methanol or methanol in combinationwith other solvents crystallizes out the less polar IVa β anomer almostcompletely with substantially no contamination of the IVa α anomer.

The coupling reaction is generally carried out at or below roomtemperature and preferably at about -10° to -40° C. While the couplingreaction proceeds slower at lower temperatures, the lower temperaturesfavor the formation of the IVa C-1"-β anomer by slowing anomerization ofIIIa in the reaction mixture. For example, the coupling reaction ofdibenzyl 4'-demethyl-4-epipodophyllotoxin-4'-phosphate and2,3-di-O-benzyl-4,6-O-ethylidene-α,β-D-glucopyranose (85:15 β:α) inacetonitrile at -20° C. produces the IVa β and IVa α in a ratio of72:28, while at -40° C., the ratio is 74:26. The same coupling reactionin propionitrile at -20° C. results in a IVa β to IVa α ratio of 57:43,while at -78° C., the ratio is 76:24.

The preferred solvent for the coupling reaction is acetonitrile sincethe reaction proceeds rapidly compared to the standard solvents forcoupling reactions. Acetonitrile has the unexpected property of enablingcoupling reaction to reach completion in about two hours, while thereaction in dichloroethane takes about 18 hours. The coupling reactionin acetonitrile is faster than propionitrile. Furthermore, the couplingreaction in acetonitrile allows greater formation of the IVa β anomer.Several solvents were studied in the coupling reaction of dibenzyl4'-demethyl-4-epipodophyllotoxin-4'-phosphate and2,3-O-benzyl-4,6-O-ethylidene-α,β-D-glucopyranose. Typically, the β:αratio increased with higher dielectric constant of the solvent.

The substituents on the substituted benzyl protecting groups on theglucopyranose also influence the ratio of formation of IVa β to IVa α.For example, bulky groups in the ortho position favor the C-1"-β form ofCompound IV by creating steric hindrance in Compound IVα while littlehindrance is caused by meta and para substituents. Electron withdrawinggroups also favored the C-1"-β anomer. The highest β:α ratio is obtainedwith pentafluorobenzyl, which produced a IVa C-1"-β to IVa C-1"-α ratioof 80:20.

The anomeric mixture of IVa α,β is separated to obtain substantiallypure C-1"-β form by a single crystallization step after standardwork-up. The anomeric mixture of IVa α,β is dissolved in methanol. Thesolution is heated to reflux to completely dissolve the compound IVaα,β. The solution is allowed to cool to room temperature. The resultingprecipitate is the substantially pure C-1"-β form of Compound IVa.

In preferred embodiments, the crystallization to obtain the C-1"-β formof Compound IVa is carried out directly with the coupling reaction.After the coupling reaction is completed and without further extractionor standard work-up, methanol is added to the solution and the solutionis allowed to warm to 0° C. The solution is then allowed to stand at 0°C. for several hours. The resulting solid has been found to besubstantially pure IVa C-1"-β.

The ability to directly crystallize the C-1"-β anomer of Compound IVaeven from a 50:50 anomeric mixture is a significant and unexpectedadvantage of the process. As reported in J. March, Advanced OrganicChemistry, 4th Ed., John Wiley and Sons, New York, 1992, p. 121, veryfew diastereomers are able to be separated by a single crystallization.

After recovery of the C-1"-β anomer of Compound IV, the hydroxy and thephosphate protecting groups are removed simultaneously by known methodsand preferably by hydrogenation. The hydrogenation deprotection stepproceeds efficiently to produce etoposide phosphate in high yields withminimal degradation. Compounds IV, IVa and IVb are very labile andsensitive to both acid and base. Existing processes of using acids orbases to remove the hydroxy and phosphate protecting groups usuallyresult in decomposition of a portion of the desired product. Inaddition, the deprotection steps can cleave the ethylidene group fromthe glucopyranose. Compared to existing processes for removing theprotecting groups, the hydrogenation process is advantageous in thatonly one deprotection step is required, no heavy metals are required,and the process is conducted under mild, neutral conditions to give ahigh yield. Chromatography is not required to obtain etoposide phosphatein pure form as in other processes.

The hydrogenation may be by a number of known processes. Typically, thehydrogenation is in the presence of a noble metal catalyst in a suitablesolvent or solvent mixture.

In preferred embodiments, the hydrogenation is carried out using 4%palladium on carbon in a solution of Compound IVa C-1"-β in 50/50methanol/tetrahydrofuran (THF). The mixture is hydrogenated for severalhours, typically 3-6 hours, at 40-50 psig hydrogen. The catalyst maythen be removed by filtering, and the etoposide phosphate recrystallizedfrom ethanol. The deprotection of IV C-1"-β to etoposide phosphate ofFormula V is as follows: ##STR20##

The 4'-demethylepipodophyllotoxin glucoside 4'-phosphate of Formula Vmay be converted to its pharmaceutically acceptable salt by contactingit with a source of the appropriate cation. For example, a sodium saltmay be made by treating the phosphate with a suitable sodium base,resulting in the formation of-the sodium salt thereof. Solvates of the4'-demethylepipodophyllotoxin glucoside 4'-phosphate of Formula V mayalso be obtained by known methods.

The etoposide phosphate may further be converted to etoposide byremoving the phosphate group using a phosphatase enzyme in an aqueousbuffer. Phosphatase is able to convert etoposide phosphate completely toetoposide. The reaction is carried out in a tank with a buffer at a pHof about 5-12 and preferably at pH 6-9 at room temperature. Typically,etoposide phosphate is in the form of a solvate when mixed with theaqueous buffer.

The enzymatic conversion of etoposide 4'-phosphate to etoposide isadvantageous since the conversion is carried out under mild conditionswithout degradation of the etoposide or etoposide 4'-phosphate. Forexample, the labile ethylidene group is substantially unaffected by thephosphatase enzyme. The enzyme may be any enzyme having phosphataseactivity at pH 5-12 and preferably pH 6-9. Suitable phosphatase enzymesinclude acid and alkaline phosphatase. The phosphatase may be obtainedfrom bovine, bacterial or other sources such as bovine and calfintestinal mucosa. Alternatively, the phosphatase may be wheat germlipase which is known to have phosphatase activity. These enzymes areavailable from Sigma Chemical Company.

Suitable buffers, for example, include M-Tris pH 7.8, M-Tris pH 8.7,M-Borate pH 10.0 and M-Bicarbonate pH 10.3. The dephosphorylation ofEtoposide phosphate V to Etoposide VI is as follows:

The following non-limiting examples demonstrate preferred embodiments ofthe invention.

EXAMPLE 1

2,3-Di-O-benzyl-4,6-O-ethylidene-α,β-D-glucopyranose (IIIa α,β). Thiscompound was prepared according to adaptation of literature proceduresfor analogous compounds as disclosed in U.S. Pat. No. 4,997,931. ¹ H NMRshowed the anomeric composition to be 57:43 β:α. R_(f) (40%EtOAc/hexane): 0.40. ¹ H NMR (CDCl₃): δ7.39-7.27 (m, 10H), 5.14 (d,0.5H, J=3.7 Hz), 4.91-4.66 (m, 5.5H), 4.14 (dd, 0.5H, J=5.0, 10.5 Hz),4.09 (dd, 0.5H, J=5.0, 10.3 Hz), 3.94-3.88 (m, 1H), 3.66 (t, 0 .5H,J=9.0 Hz), 3.56-3.25 (m, 3.5H), 3.10 (bs, 1H, conc. dependent OH), 1.36(d, 3H, J=5.0 Hz). ¹³ C NMR (CDCl₃) : δ128.53, 128.42, 128.31, 128.09,127.95, 127.83, 127.63, 99.50, 97.72, 92.12, 82.94, 81.44, 81.08, 80.89,79.31, 78.33, 75.23, 75.12, 74.96, 73.81, 68.53, 68.22, 66.22, 62.48,20.43.

EXAMPLE 2

2,3-Di-O-benzyl-4,6-O-ethylidene-β-D-glucopyranose (IIIa β). Theanomeric mixture IIIa α,β (7 g) was placed in a 250 ml roundbottomflask. Hexane (125 ml) was added, and the suspension was heated toreflux. The sugar became an insoluble oil which sank to the bottom. Thesuspension was allowed to cool to room temperature, then a stir bar wasadded and the solution was gently stirred overnight. White, fluffycrystals formed and floated in the hexane above the rest of the impuresolid. The crystals were collected by decanting the supernatant into aBuchner funnel. The impure solid was left in the flask. The white solidIIIa β was dried at room temperature under vacuum (20 mm Hg). ¹ H NMR(CDCl₃): δ7.37-7.27 (m, 10H), 4.90-4.69 (m, 6H), 4.14 (dd, 1H, J=4.9,10.4 Hz), 3.66 (t, 1H, J=9.0 Hz), 3.54 (t, 1H, J=10.2 Hz), 3.45 (t, 1H,J=9.3 Hz), 3.37-3.27 (m, 2H), 3.23 (d, 1H, J= 5.5 Hz, conc. dependentOH), 1.36 (d, 3H, J=5.1Hz). ¹³ C NMR (CDCl₃): δ128.42, 128.29, 128.11,127.93, 127.82, 127.63, 99.45, 97.71, 82.93, 81.06, 80.88, 75.22, 74.96,68.21, 66.21, 20.39.

EXAMPLE 3

Dibenzyl 4'-demethyl-4-epipodophyllotoxin-4'-phosphate (IIa). Anoven-dried, three-neck 1L roundbottom flask was fitted with a droppingfunnel, stir bar, thermometer, two septa, and N₂ inlet. The flask wascharged with 4'-demethylepipodophyllotoxin (I, 25.00 g, 62.45 mmol) andanhydrous acetonitrile (367 ml, 0.17M). The suspension was cooled to-10° C. Carbon tetrachloride (30.1 ml, 312.25 mmol) was added, keepingthe temperature at -10° C. N,N-Diisopropylethylamine (22.84 ml, 131.15mmol) was added by syringe over 3 minutes. N,N-dimethylaminopyridine(0.763 g, 6.25 mmol) was added all in one portion, followed by thedropwise addition of dibenzyl phosphite (20.00 ml, 90.55 mmol) over a 15minute period. The reaction was somewhat exothermic during the addition,but the internal temperature was kept at 10° C. with additional externalcooling. The reaction was stirred at -10° C. for 37 minutes. During thistime, the starting material dissolved and the reaction was followed byHPLC. 0.5M KH₂ PO₄ (150 ml) was added and the solution was allowed towarm to room temperature. The mixture was extracted with EtOAc (1×350ml) and then washed with water (2×100 ml). The organic layer was driedover Na₂ SO₄ and concentrated in vacuo to a volume of 150 ml. 2-Propanol(500 ml) was added. Solvent (200 ml) was removed in vacuo and solidprecipitated during this time. 2-Propanol (500 ml) was added and thenanother 550 ml of solvent was removed in vacuo. Finally, 2-propanol (250ml) was added and the mixture was heated to reflux until all soliddissolved. The yellow solution was cooled to room temperature and thento 0° C. for 4 hours. A white solid was collected, washed twice withcold 2-propanol and dried in vacuo (40° C., 20 mm Hg) to yield 37.15 g(90.1%). HPLC R_(t) R_(f) (10% MeOH/CH₂ Cl₂): 0.66. ¹ H NMR (CDCl₃):δ7.37-7.28 (m, 10H), 6.81 (s, 1H), 6.39 (s, 1H), 6.30 (s, 2H), 5.90 (dd,2H, J=l.0, 12.7 (Hz), 5.28-5.14 (m, 4 H), 4.71 (d, 1H, J=3.4 Hz), 4.53(d, 1H, J=5.1Hz), 4.25 (dd, 1H, J=8.7, 10.7 Hz), 3.63 (s, 6H), 3.27 (dd,1H, J=5.2, 14.1 Hz), 2.71-2.61 (m, 1H). ¹³ C NMR (CDCl₃): δ175.27,151.15, 151.11, 148.22, 147.32, 137.28, 136.04, 135.94, 132.19, 131.35,128.43, 128.30, 128.26, 127.69, 127.64, 110.13, 109.32, 107.66, 101.45,69.62, 69.53, 69.46, 67.75, 66.17, 56.06, 43.81, 40.39, 38.47.

EXAMPLE 4

Dibenzyl4-(2,3-di-O-benzyl-4,6-O-ethylidene-β-D-glucopyranosyl)-4'-demethyl-4-epipodophyllotoxin-4'-phosphate(IVa β) (coupling in acetonitrile). An oven-dried 25 ml two-neckroundbottom flask fitted with a stir bar, thermometer, septa, and N₂inlet was charged with dibenzyl4'-demethyl-4-epipodophyllotoxin-4'-phosphate (IIa, 1.00 g, 1.51 mmol),dry 4A molecular sieves (1/16" pellet) (2.0 g),2,3-di-O-benzyl-4,6-O-ethylidene-α,β-D-glucose (IIIa β,α, 85:15, 0.702g, 1.817 mmol), and anhydrous acetonitrile (10.0 ml). The solution wasstirred until homogeneous and then cooled to -20° C. Boron trifluorideetherate (0.50 ml, 4.08 mmol) was added dropwise over 2 minutes. Thereaction was held at -20° C. for 80 minutes. White solid beganprecipitating 45 minutes after addition of BF₃. Pyridine (5.23 ml, 64.7mmol) was added. The suspension was allowed to warm to room temperatureand was diluted with CH₂ Cl₂ (10 ml). The white solid dissolved. Thesolution was filtered to remove remaining solids. The solution waswashed with 3% HCl (7 ml), and then the aqueous phase was back extractedwith CH₂ Cl₂ (10 ml). The combined organic phase was washed with water(7 ml) and the aqueous phase was back extracted with CH₂ Cl₂ (10 ml).The combined organic phase was washed finally with saturated NaCl (7ml). The organic layer was dried over Na₂ SO₄ and concentrated in vacuoto a white/yellow solid. HPLC of the crude product showed a 71.6:28.4ratio of IVa β:IVa α. The solid was dissolved in CH₂ Cl₂ (10 ml) withstirring. Methanol (90 ml) was added. Some solid soon precipitated out.The solution was warmed to reflux with stirring, during which time thesolid dissolved, and then 20 ml of solvent was distilled off. The solidbegan crystallizing after 19 ml was collected. The mixture was allowedto cool to room temperature while stirring gently for 5 hours. The whitesolid was collected and rinsed twice with room temperature methanol. Thesolid IVa β was dried in vacuo (40° C., 20 mm Hg) and yielded 0.830 g(53.3%). R_(f) (50% EtOAc hexane): 0.36. ¹ H NMR (CDCl₃): δ7.38-7.18 (m,18 H), 7.00-6.98 (m, 2H), 6.82 (s, 1H), 6.54 (s, 1H), 6.25 (s, 2H),5.97-5.89 (dd, 2H, J=l.0, 26.7 Hz), 5.29-5.18 (m, 4H), 4.89-4.85 (m,2H), 4.77-4.71 (m, 3H), 4.60-4.49 (m, 3H), 4.39 (t, 1H, J=10.2 Hz), 4.23(t, 1H, J=8.2 Hz), 4.16 (dd, 1H, J=4.9, 10.4 Hz), 3.63 (s, 6H), 3.55 (t,1H, J=10.2 Hz), 3.45-3.34 (m, 2H), 3.32-3.21 (m, 2H), 2.89-2.80 (m, 1H),1.38 (d, 3H, J=5.0 Hz). ¹³ C NMR (CDCl₃): δ174.74, 151.20, 148.72,147.17, 138.48, 137.75, 137.0, 136.3, 136.2, 132.02, 128.62, 128.42,128.30, 128.21, 128.07, 127.87, 127.70, 127.67, 110.72, 109.18, 107.73,102.32, 101.60, 99.55, 81.66, 80.95, 75.40, 75.06, 73.45, 69.45, 68.19,67.87, 65.97, 43.87, 41.22, 37.48, 20.40.

The C-1"-α isomer IVα remained in the mother liquor, along with some ofthe desired product IVβ (IVβ:IVα 13.7:86.3).

EXAMPLE 5

Dibenzyl4-(2,3-di-O-benzyl-4,6-O-ethylidene-β-D-glucopyranosyl)-4'-demethyl-4-epipodophyllotoxin-4'-phosphate(IVa β) (coupling in dichloroethane). An oven-dried 250 ml three-neckroundbottom flask fitted with a stir bar, thermometer, two septa and N₂inlet was charged with dibenzyl4'-demethyl-4-epipodophyllotoxin-4'-phosphate (IIa, 14.295 g, 21.57mmol), dry 4A molecular sieves (1/16" pellet) (28.6 g),2,3-di-O-benzyl-4,6-O-ethylidene-α,β-D-glucose (IIIa α,β, 10.0 g, 25.88mmol), and anhydrous 1,2-dichloroethane (143 ml). The solution wasstirred until homogenous and then cooled to -20° C. Boron trifluorideetherate (7.15 ml, 58.24 mmol) was added dropwise over 10 minutes. Thereaction was held at -20° C. for 18 hours. Pyridine (5.23 ml, 64.7 mmol)was added and the mixture turned from brown to yellow. The cloudysolution was allowed to warm to room temperature and was diluted withCH₂ Cl₂ (200 ml) and filtered to remove solids. The solution was washedwith 3% HCl (100 ml), water (100 ml) and finally saturated NaCl (100ml). The organic layer was dried over Na₂ SO₄ and concentrated in vacuoto a yellow oil. Refluxing methanol (1500 ml) was added while stirring.The mixture was allowed to cool to room temperature and stand overnight.The white solid was collected and rinsed twice with methanol. The solidIVa β was dried in vacuo (40° C., 20 mm Hg) and yielded 8.86g (39.8%).

The C-1"-α isomer IVa α remained in the mother liquor, along with someof the desired product IVa β. This remaining coupled product wasrecovered by further crystallization and/or chromatography. The ratio ofβ:α of the crude product before crystallization of IVβ was 54:46. Theoverall yield of coupled product was 81%.

Dibenzyl4-(2,3-di-O-benzyl-4,6-O-ethylidene-α-D-glucopyranosyl)-4'-demethyl-4-epipodophyllotoxin-4'-phosphate(IVa α): R_(f) (50% EtOAc/hexane): 0.31. ¹ H NMR (CDCl₃): δ7.38-7.21 (m,20H), 6.87 (s, 1H), 6.26 (s, 2H), 5.95 (d, 2H, J=5.8 Hz), 5.29-5.18 (m,4H), 4.87 (dd, 3H, J=2.3, 11.1 Hz), 4.79-4.74 (m, 2H), 4.68-4.58 (m, 4H), 4.11 (t, 1H, J=7.9 Hz), 3.95 (q, 1H, J=10.6 Hz), 3.86 (t, 1H, J=9.2Hz), 3.63 (s, 6H), 3.51 (dd, 1H, J=3.6, 9.4 Hz), 3.45 (d, 1H, J=7.2 Hz),3.45-3.35 (m, 3H), 2.82-2.75 (m, 1H), 1.32 (d, 3 H, J=5.0 Hz). ¹³ C NMR(CDCl₃): δ174.91, 151.22, 151.18, 148.44, 147.02, 138.56, 137.83,137.05, 136.27, 136.18, 132.19, 129.27, 128.59, 128.45, 128.34, 128.24,128.12, 127.96, 127.89, 127.72, 127.69, 110.44, 109.81, 107.85, 101.61,101.08, 99.59, 82.07, 79.36, 78.59, 76.76, 75.09, 74.69, 69.52, 69.46,69.41, 68.18, 67.04, 62.95, 56.15, 43.82, 41.10, 38.41, 20.40.

EXAMPLE 6

This example demonstrates coupling and crystallization steps beingcarried out in the same reaction vessel. A 50 ml three-neck roundbottomflask with a stir bar was oven-dried, fitted with two septa, and cooledunder N₂. Dibenzyl 4'-demethyl-4-epipodophyllotoxin- 4'-phosphate (1.002g, 1.51 mmol) and 2,3-O-benzyl-4,6-O-ethylideneglucopyranose (IIa 85:15β:α, 0.702 g, 1.81 mmol) were added. The solids were dissolved inanhydrous acetonitrile (10.0 ml), and then the solution was cooled to-40° C. Boron trifluoride etherate (0.50 ml, 4.1 mmol) was addeddropwise. The solution was stirred at -40° C. and followed by HPLC.During the reaction, some product precipitated. After 6 hours, methanol(30 ml) was added dropwise. The suspension was allowed to warm to -30°C. with stirring, then allowed to stand at 0° C. without stirring for 17hours. The solid was collected in a Buchner funnel and rinsed twice withroom temperature methanol. This produced 0.9668 g (62.0%) of IVa β withHI (homogeneity index) of 100%.

EXAMPLE 7

Etoposide-4'-phosphate (V): 4% Palladium on carbon, 50% wet (314 mg) wasadded to a solution of dibenzyl4-(2,3-di-O-benzyl-4,6-O-ethylidene-β-D-glucopyranosyl)-4'-demethyl-4-epipodophyllotoxin-4'-phosphate(IVa β, 758 mg) in 50/50 MeOH/THF (50 ml). The mixture was hydrogenatedat ambient temperature and 40-50 psig hydrogen for 3-6 hours. Thecatalyst was filtered off and rinsed with MeOH. The filtrate wasconcentrated in vacuo (40°-60° C., aspirator) to a volume of 8-10 ml.Absolute ethanol (50 ml) was added and the solution was againconcentrated to -10 ml. Ethanol (25 ml) was again added and the solutionwas concentrated to 10 ml. Seed crystals of etoposide-4'-phosphatediethanol solvate were added and the temperature of the solution wasadjusted from about 50° C. to 15°-20° C. over 30-60 minutes. Afterholding at 15°-20° C. another 30 minutes, the white crystals werecollected by filtration and washed with 5° C. ethanol (5-10 ml). Thesolid was dried under high vacuum at 25°-40° C. There was obtained 436mg (77.8%) of etoposide-4'-phosphate diethanol solvate (V) which assayedat 99.2 area % purity by HPLC.

EXAMPLE 8

Etoposide (VI): With magnetic stirring, etoposide-4'-phosphate diethanolsolvate (V, 410 mg) was dissolved in 1.0M Tris buffer (8.0 ml). The pHwas adjusted from 8.1 to 8.7 with 1N NaOH. The solution was warmed to35° C. A solution (2.0 ml, 200 units/ml) of alkaline phosphatase (Sigma,catalog #P6774) in MilliQ water was added. Within 10 minutes, solidsprecipitated. The pH was maintained in the range 8.4-8.8 by adding 1NNaOH as needed. The reaction was followed by HPLC. After 3 hours, themixture was cooled to 10° C. for 15 minutes. The solid was collected byvacuum filtration, washed with water (5-7 ml), and dried under highvacuum (20° C.) for 18 hours. There was obtained 241 mg (76% ofetoposide (VI), 95.5 area % by HPLC.

What is claimed is:
 1. A process for preparing a compound having theFormula Va ##STR22## which comprises reacting an anomeric mixture of acompound of Formula IIIb ##STR23## wherein R₁ is an arylmethyl hydroxyprotecting group and R₂ is arylmethyl or the two R₂ groups together areC₁₋₅ alkylidene with a compound of Formula II in an organic solvent as areaction medium and in the presence of a Lewis acid at or below roomtemperature ##STR24## where R₃ is arylmethyl and where R₁, R₂ and R₃ arethe same or different to form an anomeric mixture of the compound ofFormula IVb ##STR25## and adding an alcohol to said reaction medium andcrystallizing and separating the C-1"-β form of Compound IVbsubstantially free of the α form directly from said anomeric mixture insaid reaction medium, and subsequently removing the hydroxy andphosphate protecting groups and in cases where R₂ is arylmethyl,reacting Compound IVb with a carbonyl having one to five carbon atoms oran acetal equivalent thereof.
 2. The process of claim 1, wherein R₁ andR₃ are benzyl or a benzyl substituted with one or more substituentsselected from the group consisting of C₁₋₄ alkyl, hydroxy, phenyl,benzyl, halogen, alkoxy nitro, carboxylic acids and esters thereof. 3.The process of claim 1, wherein said hydroxy and phosphate protectinggroups are removed simultaneously.
 4. The process of claim 1, whereinsaid hydroxy and phosphate protecting groups are simultaneously removedby hydrogenating at ambient temperature in the presence of a noble metalcatalyst.
 5. The process of claim 4, wherein said noble metal catalystis palladium.
 6. The process of claim 1, wherein said alcohol ismethanol.
 7. The process of claim 1, wherein said Lewis acid is borontrifluoride etherate.
 8. The process of claim 1, comprising reactingCompound II with Compound IIIb in a halogenated or non-halogenatedsolvent.
 9. The process of claim 8, wherein said solvent isacetonitrile.
 10. The process of claim 9, wherein said solvent containsa metal perchlorate to increase the ionic strength of the solvent. 11.The process of claim 1, further comprising preparing said compound ofFormula II by reacting a phenol of Formula I ##STR26## with adi(arylmethyl)phosphite, tetrahalomethane, trialkylamine and anacylation catalyst to form the protected phosphate of Formula II. 12.The process of claim 1, wherein said reaction is carried out at aboutroom temperature to -40° C.
 13. A compound having the Formula IV##STR27## wherein R₁ is a benzyl or substituted benzyl hydroxyprotecting group and R₃ is a benzyl or substituted benzyl phosphateprotecting group.
 14. The compound of claim 13, wherein R₁ and R₃ are abenzyl substituted with one or more selected from the group consistingof C₁₋₄ alkyl, hydroxy, phenyl, benzyl, halogen, alkoxy, nitro,carboxylic acids and esters thereof.
 15. A process of obtaining theC-1"-β anomer of Formula IVa ##STR28## said process comprising forming asolution of an anomeric mixture of a compound of Formula IV in anorganic solvent ##STR29## where R₁ and R₃ are benzyl, adding an alcoholto said solvent and crystallizing said C-1"-β anomer from said anomericmixture in said solution and recovering said crystals of the C-1"-βanomer of Formula IVa substantially free of the α anomer from saidanomeric mixture.
 16. The process of claim 15 wherein said solvent isacetonitrile.
 17. The process of claim 15, wherein said solvent is analcohol and said process comprises heating said alcohol to reflux todissolve said anomeric mixture, and cooling said solution to crystallizesaid C-1"-β anomer of Formula IVa.
 18. The process of claim 15 whereinsaid alcohol is methanol.
 19. The process of claim 4, wherein saidhydroxy and phosphate protecting groups are removed by hydrogenating atambient temperature in a 50/50 methanol-tetrahydrofuran solvent in thepresence of a 4% palladium on carbon catalyst by contacting compound IVbwith hydrogen at 40-50 psig.
 20. The process of claim 1, furthercomprising adding methanol to said anomeric mixture of the compound ofFormula IVb to dissolve said compound of Formula IVb, heating theresulting solution to reflux, and cooling said solution to roomtemperature to precipitate and separate said C-1"-β anomer of compoundIVb from the solution of said anomeric mixture.
 21. The process of claim1, comprising reacting compounds IIIb and II at about room temperatureto -40° C., and wherein said crystallizing step comprises addingmethanol to said reaction medium and warming said reaction medium toabout 0° C. to precipitate and separate said C-1"-β anomer of CompoundIVb from said anomeric mixture.
 22. The process of claim 1, comprisingreacting compounds IIIb and II in acetonitrile at about room temperatureto -40° C. in the presence of boron trifluoride etherate.
 23. Theprocess of claim 22, comprising reacting about 1.2 mole of compound IIIbper mole of compound II.
 24. The process of claim 11, comprisingreacting compound I with dibenzyl phosphite, carbon tetrachloride, N,Ndimethylaminopyridine at about -10° C. in acetonitrile, the carbontetrachloride being present in the amount of 1 or more equivalents perequivalent of compound I.
 25. The process of claim 24, comprisingreacting compound I with about 1.4 moles of dibenzyl phosphite, 5 molesof carbon tetrachloride, 2.1 moles of N,N diisopropylethylamine and 0.1mole of N,N -dimethylaminopyridine per mole of Compound I.
 26. Theprocess of claim 12, comprising reacting compound II and IIIb at about-10° C.
 27. The process of claim 15, comprising forming a solution of ananomeric mixture of compound IVa in acetonitrile, adding methanol tosaid solution, heating to reflux and cooling said solution tocrystallize and separate said C-1"-β anomer of compound IVa from thesolution of said anomeric mixture.
 28. A process for preparing acompound having the Formula V ##STR30## which comprises reacting ananomeric mixture of a compound of Formula IIIa ##STR31## with a compoundof Formula II in an organic solvent reaction medium and in the presenceof a Lewis acid selected from the group consisting of borontrifluoroetherate, AlCl₃, ZnCl₂, Et₂ AlCl, CF₃ SO₃ H, CF₃ SO₃ Ag, Zn(CF₃SO₃)₂ and TMSCF₃ SO₃ at or below room temperature ##STR32## to form ananomeric mixture of the compound of Formula IVa ##STR33## adding analcohol to said reaction medium and crystallizing and separating theC-1"-β form of Compound IVa from a solution of said anomeric mixture,and subsequently removing the benzyl protecting groups.
 29. The processof claim 28, wherein said reaction medium is acetonitrile, and saidprocess comprises reacting compounds IIIa and II at about roomtemperature to -40° C.
 30. The process of claim 28, wherein afterreacting compounds II and IIIa, said process comprising adding a solventto the resulting anomeric reaction mixture, heating the anomeric mixtureto dissolve the compound IVa and cooling the mixture to crystallize andSeparate the C-1"-β form of compound IVa from said anomeric mixture,wherein said solvent is selected from the group consisting of methanoland a mixture of methylene chloride and methanol.
 31. The process ofclaim 30, comprising heating said anomeric mixture to reflux to dissolvesaid compound IVa and cooling to room temperature to crystallize andseparate said C-1"-β form of compound IVa from said anomeric mixture.32. The process of claim 28, wherein after reacting compounds II andIIIa, adding methanol to the resulting anomeric reaction mixture todissolve the compound IVa and crystallizing and separating said C-1"-βform of compound IVa from the anomeric mixture.
 33. The process of claim28, wherein said step of removing the benzyl protecting groups compriseshydrogenating at ambient temperature in a 50/50 methanol tetrahydrofuransolvent in the presence of a 4% palladium on carbon catalyst bycontacting compound IVa with hydrogen at 40-50 psig.